1. Field of the Invention
The present invention relates to a biosensor capable of rapidly quantifying a specific component in a sample solution with high accuracy in a simplified manner, and to a method for producing the same.
2. Description of the Related Art
Various types of biosensor have heretofore been proposed as a system for quantifying the specific component in the sample solution without requiring diluting or stirring of the sample solution.
As an example of such biosensors, a glucose Sensor will be described in the following paragraphs. In general, a system combining glucose oxidase with an enzyme electrode or a hydrogen peroxide electrode is already known as a method of quantifying glucose utilizing the enzyme electrode. The glucose oxidase selectively oxidizes a substrate, i.e., .beta.-D-glucose into D-glucono-.delta.-lactone by using oxygen as an electron acceptor. During this reaction, oxygen is reduced into hydrogen peroxide. By measuring the amount of the oxygen consumed in this reaction by an oxygen electrode, or by measuring the amount of the hydrogen peroxide produced in this reaction by a hydrogen peroxide electrode which utilizes a platinum electrode or the like, the glucose in the sample solution can be quantified.
By the above-mentioned method, the measurement is however adversely influenced with a concentration of the dissolved oxygen depending on the subject of the measurement. Further, the measurement is made completely impossible under a condition lacking oxygen. A type of the glucose sensor that does not use oxygen as the electron acceptor but uses a metal complex or an organic compound such as potassium ferricyanide, a derivative of ferrocene or a derivative of quinone as the electron acceptor has therefore been developed. With this type of biosensor, by oxidizing a reductant of the electron acceptor produced as the result of the enzyme reaction by the electrode, the concentration of the glucose can be determined based on the current consumed for this oxidation reaction. This manner of measurement is not limited to glucose but has been widely applied for the quantification of substrates other than glucose.
As an example of this type of biosensor, a glucose sensor is known (Japanese Laid-Open Patent Publication No. Hei 1-114,747) which will be described below.
The disclosed biosensor has a configuration comprising an electrical insulating base provided with an electrode system including a working electrode and a counter electrode, a filter layer composed of polycarbonate porous film, an electron acceptor carrying layer, an enzyme carrying layer, a buffer carrying layer, and a developing layer composed of woven cellulose, which are sequentially laminated on the insulating base by placing some space from the electrode system. In this configuration, the above-mentioned carrying layers are prepared by impregnating cellulosic porous films with aqueous solutions of the electron acceptor, the enzyme, and the buffer, and then drying the impregnated bodies.
The operation of this glucose sensor is as follows.
The sample solution titrated on the developing layer is first passed to the buffer carrying layer, whereby the pH value of the sample solution is adjusted to a pH value that can give the highest activity to the enzyme by the buffering action of the buffer. Next, the glucose in the sample solution reacts specifically with the glucose oxidase in the enzyme carrying layer. At the same time, the electron acceptor, such as potassium ferricyanide in the electron acceptor carrying layer, is reduced by the electron produced by the above-mentioned reaction to produce potassium ferrocyanide. The amount of the produced potassium ferrocyanide is directly proportional to the concentration of glucose contained in the sample solution. After the substances having a large molecular weight such as protein which disturb the electrode reaction contained in the sample solution are filtered off by the filter layer, the sample solution reaches the electrode system provided on the insulating base. In order to prevent erroneous measurement, part of the electrode system is covered with the insulating layer. By measuring the value of the current for oxidizing the potassium ferrocyanide produced in the sample solution by the electrode system it is possible to determine the glucose concentration of the sample solution.
the configuration of such prior art sensors, however, there is an inconvenience that an adverse influence is given to the responsive current, because wetting of the surface of the insulating base including the electrode system with the sample solution is not necessarily uniform and thus bubbles are retained between the porous body of the filter layer and the insulating base. Further, if the sample solution contains substances liable to be absorbed in the electrode or substances having an electrode activity, there would be a case wherein the response of the sensor is adversely influenced.
As a method for overcoming the above-mentioned inconveniences, the following biosensor is proposed and disclosed in Japanese Laid-Open Patent Publication No. Hei 2-062,952.
In the disclosed configuration, the sensor comprises an electrically insulating base, an electrode system composed of a working electrode, a counter electrode and a reference electrode formed on the insulating base by means of screen printing or the like, and a reaction layer including a hydrophilic polymer, an oxido-reductase, an electron acceptor, and a buffer as well if required, formed on the electrode system in a manner that the reaction layer is in close contact with the electrode system.
When the sample solution containing the substrate is titrated on the reaction layer, the reaction layer dissolves in the sample solution which is thereby adjusted to a pH value at which the highest enzyme activity is achieved by the buffering action of the buffer, the enzyme reacts with the substrate, and the electron acceptor is reduced. After the completion of the enzyme reaction, the reduced electron acceptor is electrochemically oxidized, and the concentration of the substrate contained in the sample solution is derived from the value Of the current consumed for oxidizing the electron acceptor.
In the above-mentioned configuration of the prior art sensor, if the biosensor is moistened, the buffer would be partly mixed with the enzyme to induce a chemical interaction, thereby lowering the enzyme activity and deteriorating the storing property of the biosensor.